Host adaptation of retroviral vectors

ABSTRACT

A method of making retrovirus vectors having selected characteristics, in particular an increased ability to infect a particular target cell, comprises subjecting a starting retrovirus or retroviral vector to a selection process in vitro which involves a plurality of rounds of infection of a host cell during which the retrovirus or retroviral vector evolves to attain the selected characteristics. Components of the evolved retrovirus or retroviral vector can be used in retroviral vector production systems for producing retroviral vectors having the selected characteristics. The invention is particularly useful for preparing retroviral vectors suitable for gene therapy.

This invention relates to improvements in retroviral vectors for genetherapy and other uses. In particular the invention relates to methodsfor producing improved retroviral vectors, and for producing improvedretroviruses which may be used to make retroviral vectors. The inventionfurther relates to the retroviruses and retroviral vectors produced bysuch methods and to retroviral production systems and packaging celllines derived using the retroviruses and retroviral vectors.

Within the field of gene therapy retroviral vectors are the most widelyused gene delivery system. The most commonly used retroviral vectors arebased on murine leukaemia virus (MLV). Furthermore, MLV-based vectorsare the only retroviral vectors that have been used in human clinicaltrials (Morgan & Anderson 1993). MLV is a simple C-type retrovirus, themolecular biology of which has been reviewed extensively. The commonlyused vectors are usually produced using packaging cells lines containingintegrated copies of a gag-pol expression cassette and an env expressioncassette (McClaughlin et al 1990). A plasmid encoding the vector genome(RNA) is transfected into the packaging cell to produce the RNA speciesthat is packaged into the viral particles encoded by gag-pol and env.The retroviral particles so produced generally have one of two distincttropisms. If the env gene in the packaging cell encodes envelopeproteins from an ecotropic virus then the retroviral vector willtransduce only murine and rat cells. If the env gene is from anamphotropic virus then the vector will transduce a broader range of celltypes including human cells. The determinants of tropism residesubstantially, therefore, in the envelope protein(s) (Hunter &Swanstrom, 1990). In particular the variable regions known as VRA andVRB determine whether the surface protein (SU, gp70) binds the ecotropicor amphotropic receptors (Baftini et al, 1992). The ecotropic receptoris the murine basic amino acid transporter (Albritton et al 1989) andthe amphotropic receptor is a widely distributed phosphate transporter(Miller et al 1994; VanZeijl et al, 1994). In order to transduce humancells for therapeutic purposes amphotropic vectors are used.

Amphotropic vectors are derived from natural amphotropic MLV variants,isolated in 1976, that are capable of infecting a wide range of celltypes (Rasheed et al., 1976; Hartley and Rowe, 1976). These vectors werefirst produced in the mid-1980s (e.g. Cone and Mulligan, 1984) usingcomponents from the original viruses. Although amphotropic vectorstransduce human cells these vectors are generally an order of magnitudeless efficient at transducing human cell lines compared with theirefficiency on murine cells. Also, even though they are being used widelyfor gene therapy trials in man the efficiency of transduction is veryvariable from primary cell type to cell type (Rosenberg et al., 1990;Grossman et al., 1994) and this seriously limits the usefulness of thesesystems. The origin of this variability is not known.

There is a need for retroviral vectors which are more efficient attransducing human cells. There is also a need for retroviral vectorswhich are more suited than existing vectors for gene therapy in humansor other uses of retroviral vectors such as induction of disease mimicsin animals. The invention addresses these needs.

The invention provides in one aspect a method of making a retroviralvector having one or more selected characteristics, which methodcomprises:

(i) providing a starting retrovirus or retroviral vector, and a hostcell for the retrovirus or the retroviral vector;

(ii) subjecting the starting retrovirus or retroviral vector to aselection process in vitro which selection process involves a pluralityof rounds of infection of the host cell during which the retrovirus orretroviral vector evolves to attain the selected characteristic orcharacteristics; and

(iii) where a starting retrovirus is provided in (i), using theretrovirus resulting from (ii) in at least one component of a retroviralvector production system for producing retroviral vectors having theselected characteristic or characteristics.

Thus, by a process which may generally be described as evolution invitro, a retroviral vector can be produced which has characteristicsmore suited to a chosen purpose. A selected characteristic according tothe invention will be associated with the genome of the retrovirus orretroviral vector resulting from the selection process (ii). Thestarting retrovirus or retrovirus vector will generally be a populationof retrovirus or retrovirus vectors, which population evolves in vitroin the method according to the invention.

In another aspect, the invention provides retroviral vectors made by amethod as described herein.

In a further aspect the invention provides a retroviral vectorproduction system, said system having at least one component associatedwith a selected characteristic or characteristics attained by a methodas described herein and transferred from the retrovirus or retroviralvector into the retroviral vector production system.

The retroviral vector production system preferably comprises a packagingcell line transfected with a DNA construct encoding a packagableretroviral vector genome. The selected characteristic or characteristicsmay be associated with the vector genome or with one or more componentsof the packaging cell line, or with both.

In still further aspects, the invention provides expression vectorscomprising components of retroviral vector production systems, whichcomponents are derived from retroviruses or retroviral vectors resultingfrom a selection process as described herein. The invention alsoprovides methods of making retroviral vector production systems whichsystems comprise such components.

The starting retrovirus or retroviral vector may be derived from anysuitable retrovirus or retroviruses. MLV is presently the mostextensively studied retrovirus in this field and MLV-based vectors havebeen used in gene therapy. The invention is not however limited toMLV-based vectors. Suitable retroviruses include other oncoretroviruses(the sub-group of retroviruses of which MLV is a member), orlentiviruses (which include HIV, SIV, FIV, BLV, EIAV, CEV and visnavirus), or retroviruses from other sub-groups.

The term retroviral vector is used here to describe a defectiveretrovirus which is not by itself replication competent. However, it iscapable of infection of a host cell, and thus has a genome which iscapable of integrating into the host cell genome and has a packagingsignal. Thus, a retroviral vector usually lacks at least one of thepackaging components gag-pol, and env. When a starting retroviral vectoris used in the method according to the invention, the host cell willneed to contain the missing packaging component or components in orderthat infection of the host cell by the vector may result in theproduction of further retroviral vector particles.

A starting retrovirus contains functional env and gag-pol in its genomeand is replication competent. Even so, a starting retrovirus is notnecessarily a wild type retrovirus; generally it will have been modifiedin some way while still remaining replication competent. The use ofmodified retroviruses is preferred because these can bewell-characterised and designed to meet certain needs. For example, forpractical reasons it may be necessary to include a selectable marker inthe genome of the starting virus, so that infected host cells can beselected for. Also, it may be preferable for the virus genome to beunder the transcriptional control of a high efficiency promoter such asthe cytomegalovirus (CMV) promoter, in place of the viral LTR U3promoter. The purpose of this would be to ensure that viral genomeproduction in infected cells is not a rate-limiting step during theselection process, so that a characteristic such as more efficientinfection can be effectively selected for. These features of the genomemay be employed in the genome of a starting retroviral vector as well asa starting retrovirus.

Selection conditions are imposed in the method of the inventionaccording to the characteristic or characteristics it is desired toachieve. A preferred characteristic for selection according to theinvention is an improved ability to infect a particular target celltype. However, the invention is not limited to characteristics relatingto infection. Other desired characteristics may be selected for such asan improved ability to is withstand harsh conditions, either chemical orphysical. Selection processes for characteristics such as these are alsodescribed in detail herein.

The basic steps of infection of a target cell by a retrovirus orretroviral vector include attachment to the target cell and entry intothe cell via surface proteins in particular the envelope protein encodedby env, unpackaging of the RNA genome, reverse transcription of thegenome to produce a double-stranded DNA version or provirus, migrationto the nucleus and integration of the DNA into the target cell genome.The gene products of gag-pol are also involved in the infection process.Transduction is a term used to describe this process for retroviralvectors and for replication competent retroviruses carrying heterologousgenetic material in the genome. Generally, transduction is used to referto the process of introducing a foreign gene, particularly atherapeutically active gene, into a target cell by means of a retroviralvector.

Thus, improved infection of a target cell may be associated with env orwith gag-pol, or it may be associated with a part of the viral or vectorgenome which is not env or gag-pol, or it may be associated with acombination of any or all of these.

In particular for the purpose of gene therapy, there is a need. forretroviral vectors which are better able to infect certain cell typesthan existing vectors. Such cell types include for example human primaryblood lymphocytes and cancer cells. When generating virus or vectorsbetter able to infect a chosen cell type, it will be preferable to do sousing host cells which are as similar as possible to the chosen celltype. For practical reasons, the host cells will generally be from acell line, which may be derived from primary cells of the chosen celltype. Alternatively, it may be possible to use a supply of primary cellsof the chosen cell type. One possible strategy is to use a two stageselection process, evolving the virus or vector first on a cell line andthen on fresh primary cells.

The retroviral vectors resulting from the selection process in themethod according to the invention may be immediately useful inapplications such as gene therapy. Usually however modifications willfirst of all be required. Typically, the retrovirus or retroviral vectorresulting from the selection process of step (ii) of the methodaccording to the invention is used to provide one or more components ofa retroviral vector production system for producing retroviral vectorsuseful for gene therapy or other medical applications. A retroviralvector production system requires packaging components for packaging asuitable vector genome. Depending on the characteristics selected for,the retrovirus or retroviral vector may be used to contribute to thepackaging components or the genome or both.

In one particular embodiment of the method according to the invention astarting retroviral vector comprising the env gene and a genome having aselectable marker is subjected to a selection process designed toachieve more efficient infection of a particular target cell. The envgene from the retroviral vector which emerges after the selectionprocess may then be transferred into a packaging cell line which canthen be used to produce retroviral vectors having the selectedcharacteristic and suitable for use in a desired application. Theretroviral vectors produced by the packaging cell do not carry the envgene, but comprise envelope proteins encoded by the env gene.

In another embodiment, a starting retrovirus having a selectable markeris used. Following a process of selection during which the retrovirusevolves to become more efficient at infecting a chosen target cell, theenv and gag-pol genes of the virus which emerges are transferred intoexpression vectors for use in a retroviral vector production system.

A retroviral vector production system as referred to herein comprisespackaging components, that is a set of nucleic acid sequences whichencode the packaging proteins need to package a defective retroviralvector genome. When a nucleic acid sequence encoding a compatiblepackagable RNA genome is introduced into the system, retroviral vectorsmay be produced by the system. The packaging components are maintainedin cells into which they have been either transiently or stablytransfected. Most commonly these are cell lines in which the packagingcomponents are stably maintained.

It is a particular aim of this invention to provide new retroviralvector systems derived from new variants of amphotropic and ecotropicviruses produced by in vitro evolution. These variants are selected fortheir ability to infect specific target cell types or to have specificproperties that are desirable. Selection protocols vary with differentdesired end-points but several general principles are taken intoaccount.

1. Varying the mutation rate.

One of the most powerful optimisation procedures in physics/computing is“simulated annealing” (Kirkpatrick S et al, Science 220, 671,1983,Gillespie D T, J Comp Phys 22, 403, 1976). Varying temperaturegradients are used to aid the search for the global minimum and avoidthe chance of being trapped in a local minimum. For our system,temperature is equivalent to mutation rate. Alternating the mutationrate in serial passages could greatly improve the chances of findingoptimal variants. (For a theoretical description see Fontana W et al,Phys Rev A 40, 3301, 1989.) The idea is to use a very high mutation ratein one culture, in order to produce many mutants and subsequently to usea very low mutation rate in the next culture, in order to provide achance for the best variants to outgrow the others without accumulatingtoo many additional mutations.

2. Optimal mutation rate.

As far as possible the error rate of the MLV reverse transcriptaseshould be increased to maximise the production of advantageous mutationsduring the high mutation rate stages. In order that selection worksefficiently it is essential to have enough genetic variation. Ifmutation rates are too low advantageous variants may not appear fastenough and the in-vitro evolution will take too long. On the other hand,for very high mutation rates an advantageous mutation may often beaccompanied by a deleterious mutation somewhere else in the genome. Inbetween there is an optimal mutation rate. A very rough estimate showsthat the optimal mutation rate is of the order of 1 over the length ofthe genome (number of bases) (Nowak 1990, Nature 347,522). For a genomeof the size of MLV the optimal mutation rate is approximately 10⁻³-10⁻⁴.

3. Controlling the mutation rate in vitro.

Given the considerations above it is important to be able to varymutation rate during the evolution protocols so that the system goesthrough alternating rounds of generation of diversity and selection.Adding high concentrations of nucleosides or sub-inhibitoryconcentrations of nucleoside analogues to producer cell culturesincreases the error rate of reverse transcriptase (e.g. Pathak and Temin1992 J. Virol. 66, 3093). This technique is used, therefore, to alterthe mutation rate during the selection protocols. Mutation rates can bemeasured by the procedures described by Pathak and Temin (1992) inexperiments that are run in parallel with the selection protocols.

4. Restricting selection to a specific part of the life-cycle of thevirus.

In some cases it may be important to evolve components of the vectorselectively. Essentially we want to find a variant that is well adaptedto infect human cells rather than a variant that is capable of higherlevel production. Thus we want to maximise the rate and efficiency ofthe first half of the life-cycle, that is infection, including virusentry, unpacking, reverse transcripton, migration to the nucleus, andintegration. By straightforward selection for fast replicating strainswe do not differentiate between applying selection pressure to improveinfection or improve virus production from infected cells. We can adopt,therefore, various strategies to separate infection from production inthe evolution protocol.

i. Infection pulse. In this strategy we would allow infection to occurfor only a short time by adding a retrovirus inhibitor eg AZT or otherreverse transcriptase inhibitor to inhibit further infection. Thisprocedure would select for those viruses that could infect rapidly.

ii. Component specific evolution. In this system each of the threecomponents, gag-pol, env and genome, of the retroviral vector can beevolved separately in a selection protocol that substantially reducesthe probability of selecting for increased virus production in thecontext of the whole virus. For example, if one wishes to select envvariants that increase the infection of a particular target cell line asystem such as that shown in FIG. 1 may be used. The key component inthe system shown in FIG. 1 is the vector genome which contains both theenv gene that is to be the subject of the evolution procedure and aselectable marker such as the neo gene. First a virus vector populationis generated by transfecting 293T, or other cells, with a plasmidcontaining the proviral version of this vector genome. The cells containa gag-pol expression cassette. The vector genome is transientlyexpressed and packaged into virus vector particles that can be recoveredin the cell culture supernatant. This population of particles is thenused to start the rounds of diversity generation and selection. Targetcells, again expressing a gag-pol cassette, are transduced andtransductants are selected on G418. This step is both a diversitygeneration step and a selection step. Diversity is generated because ofthe error-prone reverse transcription step necessary for integration andselection for envelope ‘efficiency’ is achieved because receptor bindingand entry is essential for transfer of the neo gene. The G418^(r)population of transduced cells is then used to produce more virus vectorparticles and these are used to transduce a fresh batch of target cellsfor second round of selection. This can go on for many rounds until theefficiency of the transduction is seen to increase. At this stage theenvelope component can be separated from the genome and used in a‘conventional’ vector production system. The resulting new vectorproduction system has an env gene component that is now optimised forinfection/transduction of the target cells. Similar strategies can beused for the genome alone to optimise packaging, for example, and forgag-pol to optimise particle formation, reverse transcription andintegration.

5. Time schedule for serial transfer.

It might be essential to keep the virus in exponential growth phase.This means that there should always be enough infectable cells in theculture. In other words sampling should occur early on in the infectionselection process.

6. Recombination

Running several lines in parallel (e.g. Schober A et al, BioTechniques18, 4, 1995) and ocassionally recombining them should greatly enhancethe efficacy of the search process for optimally adapted viruses. Thetheory of genetic algorithms (Holland J H, Adaptation in natural andartificial systems, Ann Arbor: Univ of Michigan Press, 1975) suggeststhat under specific circumstances recombination leads to betteroptimization procedures (Kauffman S, The origins of order, OUP, 1993).Recombination may be achieved in vivo by using virus derived fromseveral selection strategies in a single selection scheme orrecombination in vitro may be achieved using PCR.

7. Computer models

All diversity/selection systems can be modelled on a computer to give anestimate of rates and probabilities of achieving specific evolutionaryendpoints. We refer to this as CATORV (computer aided target optimizedretroviral vector). The simulations are based on quasi-species theory(Eigen M, Schuster P, The Hypercycle, Springer, Berlin 1979, Eigen M,McCaskill J, Schuster P, J Phys Chem, 92, 6881, 1988) using conceptslike sequence space and fitness landscape.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated in the attached figures in which:

FIG. 1 shows the principle of selective evolution of a retroviralcomponent;

FIG. 2 shows a plasmid designated pMoC(4070A) containing an amphotropicMLV provirus;

FIG. 3 shows a scheme for the construction of the plasmid shown in FIG.2;

FIG. 4(parts a-c) shows retroviral vector plasmids derived fromretroviruses selected by evolution in vitro.

FIGS. 5 to 7 show viral titres obtained in the viral evolution methoddescribed in Example 2.

Figure Legends

FIG. 1—A plasmid containing a retroviral vector genome containing an envgene and a neo^(r) gene is transiently expressed in a producer cell suchas 293T, containing the necessary packaging components. Retroviralvector particles are produced and are contacted with a chosen host cellfor successive rounds of infection. Infected host cells are selected forusing G418^(R).

FIG. 2—pMoC(4070A) is a derivative of pMo(4070A) (Ott et al 1992) inwhich the U3 regions of the LTRs contain the CMV-IE enhancer.

FIG. 3—Construction of pMoC(4070A) is carried out as follows:

1) Double stranded oligonucleotide was synthesised to span the SAC1 siteat −13 in the CMV promoter to the KPN1 site at +36 in the MLV R regionsuch that +1 for the CMV promoter and +1 for the MLV R region coincide(Shinnick et al, 1981, Nature 293, 543). This was inserted into pSP46(Ogden et al., 1986, MCB 6, 4335)

2) Remaining CMV promoter sequences restored by inserting 530 bpXBAi-SACI fragment from pKV461 (Adams et al., 1988, NAR 16, 4287) intothe pSP46 plasmid containing the fragment described in 1)

3) The PVUI-KPNI fragment from above containing the CMV promoterreplaces the corresponding sequence of pLNSX (Miller and Rosman, 1989,Biotechniques 7, 980) to create pRV104.

4) Replace PVUI-KPNI fragment in pMo(4070A) with PVUI-KPNI fragment frompRV104.

5) Excise XBAI-ECORI fragment from pRV104 and add NHEI linkers. Cut withKPNI to produce the fragment shown.

6) Insert this fragment into pMo(4070A)-5′CTR cut with NHEi and KPNI.Partial digests are required. This gives the plasmid shown.

FIG. 4—A general scheme showing retroviral vector plasmids which may bederived from retroviruses selected by evolution in vitro. They may befor example a) pHITIII, or derived from a selected genome; b) pRV-VI-51;c) pRV-VI-52, all of which are described in Example 1.

The invention will now be further described in the examples whichfollow.

EXAMPLES Example 1

A major goal in gene therapy is the transduction of human primary bloodlymphocytes (PBLs) for the treatment of inherited diseases such as SCID,acquired diseases such as leukaemia and infectious diseases such asAIDS. Although, amphotropic vector preparations can be produced readilywith titres of 10⁶-10⁷ on NIH 3T3 or HeLa cells these same preparationstransduce PBLs at efficencies 34 orders of magnitude lower. An MLVvector derivative with a high transduction efficiency is needed urgentlytherefore.

As a first step towards this goal, a replication competent amphotropicMLV provirus is needed. We have used a derivative of a constructiondesignated Mo(4070A) described by Ott et al. (1992). This is a chimericgenome in which the amphotropic envelope gene from virus isolate 4070A(Rasheed et al., 1976) replaces the ecotropic envelope gene in a Mo-MLVbackbone. This provirus, together with some genomic flanking sequencesis present in a pSV2neo plasmid the whole molecule being designatedpMo(4070A). The derivative that we have produced, designated pMoC(4070A)contains the CMV-IE promoter in place of the MLV promoter in the U3region of both LTRs (FIG. 2). pMoC(4070A) was constructed according tothe scheme shown in FIG. 3 although other strategies could be used toproduce the same molecule or to produce molecules with other efficientpromoters in the U3 regions. This derivative was produced in order toincrease the production of genomes in human cells thereby achievinglarger populations and therefore greater diversity.

In addition, by ensuring that genome production is not limiting wereduce the probability of selecting variants that simply have a greatercapacity for production. Any amphotropic provirus in any plasmid wouldbe equally suitable but it is likely that enhanced expression from theLTRs would be required to generate the population sizes necessary forthe evolution strategies.

Plasmid pMoC(4070A) is used to transfect 293T cells to produce a primaryvirus stock. As an index of the virus concentration in this stockreverse transcriptase (RT) levels are measured and compared with thoseof a vector preparation of known transducing titre. Levels of reversetranscriptase are typically 100-200 units/ml which corresponds to atransducing titre of about 10⁶10⁷ cfu/ml. This represents the startingmaster stock to be used for the selection process and as a referencestock against which new variants are compared.

One milliliter of this stock is used to infect 5×10⁵ HeLa cells and5×10⁵ PBLs. Two hours later the medium is changed and 6 days later RTlevels are measured. Typically the levels from virus plated on PBLs is34 orders of magnitude lower than with HeLa cells. This represents thedifference in plating efficiency of the two cell types. The PBLs aremaintained for 20 days in order to allow replication and selection tooccur and then 5ml supernatant from the PBLs is plated on 5×10⁵ HeLacells to amplify the population over a period of a few days until thesupernatant RT levels are equivalent to a particle titre of about10⁶/ml. 2 ml of this HeLa supernatant is plated on 5×10⁵ PBLs and thePBLs are maintained for 20 days to allow further selection to occur.During both the amplification stage on HeLa cells and the selection onPBLs the infection is monitored every 4 days by immunofluorescence usinganti-env antibody and RT to detect any emerging virus. This cycle ofPBL-Hela-PBL is designated one round of selection.

After several rounds of selection 1 ml of the virus supernatant isplated on PBLs and HeLa and 2 hours later the medium is changed and 6days afterwards RT levels are measured. A reduction of the differencebetween the two cell types shows that an MLV variant is emerging withincreased infectivity for PBLs. This strategy is employed in 10 paralleltracks of selection in order to produce 10 different selectedpopulations. These new populations presumably comprising variants withincreased replication capability on PBLs are designated MLVP^(PBL1-10).These are then combined and several rounds of selection are carried outwith the mixed virus populations at high multiplicities of infection inorder to facilitate recombination and competition. At the end a virusmaximally adapted to growth on PBLs emerges. This virus is designatedMLV^(PBL-V1).

MLV^(PBL-V1) is then used to produce plasmids for helper-free vectorproduction following standard procedures described in papers such asSoneoka et al. (1995) and Miller and Rossman (1989) and referencestherein. These have the characteristics of the plasmids shown in FIG. 4.These are designated pRV-V1-51 (gag-pol) and pRV-V1-52 (env). These twoplasmids are then used to transduce 293T cells with pHIT111 (a lacZcarrying vector genome plasmid) (Soneoka et al., 1995) to produce avector stock which is titred on NIH 3T3 cells and HeLa cells and thenused to transduce PBLs. The PBLs are then stained for expression ofB-galactosidase. A similar experiment is carried out with a standardamphotropic vector system and the numbers of blue cells compared. Thevector produced from the MLV^(PBL-V1) components gives substantiallygreater numbers of blue PBLs demonstrating that the new vector systemhas increased transducing potential for PBLs. This vector system will beof greater use therefore in gene therapy protocols requiring genetransfer into PBLs.

We have called these new vectors Target Optimised Retroviral Vectors(TORVs)

Example 2

MLV Variant Adapted for Infection of Human Ovarian Cancer Cells (SW626)

The producer cell line 293T was transiently transfected with 5 μg ofpMoC(4070A) using the overnight calcium phosphate method described byGorman et al. (1985). Viral supernatant was harvested 48 hourspost-transfection and filtered using a 0.45μm filter (Sartorius). MLV RTlevels (Goff et al. 1981) were converted to the viral titre from astandard curve relating RT activity to viral titre.

The target cancer cell line SW626 was plated at a density of 1.0×10⁶cells in a 75 cm flask and infected with 3 mls of filtered viralsupernatant in a total of 5 mls of complete medium containing 8 μg/ml ofpolybrene. Twelve hours later fresh medium was added to the infectedcells. Once the cells had reached 90% confluency the cells were split1:5. Samples of virus supematant were collected routinely throughout theexperiment and stored at −70° C. The growth pattern of the virusMOLV4040A in SW626 cells was followed over a 25 day period. The infectedcells were split 5 times during this time period on days 3, 5, 11, 16and 22 and triplicate viral supernatant samples were taken on days 3, 5,8, 9, 11, 12, 15, 16, 17, 22 and 25. It should be noted that no new(uninfected) cells were added to the system during this time period.Cells were monitored continuously for phenotypic changes to determinewhether the virus had a detrimental effect on them. The viral growthpattern of MOLV4070A in SW626 cells was analysed by MLV RT assays andviral titres were determined (FIG. 5) as described before. Virusproduction in SW626 cells produced titres up to 1.5×10⁴ on days 10 and20. The peaks and troughs of viral titre observed from the infectedSW626 cells reflects the splitting of these. However it was clear fromthese data that a chronically infected cell line had been established. Asample of virus was stored at this time and designated MLV626-0.

Virus from SW626 cells chronically infected with MOLV4070A was used toinfect new uninfected cells which were plated out at a density of 2×10⁶in a 75 cm flask. Triplicate viral supernatant samples were taken every2 to 3 days. Once the cells had reached 90% confluency the virussupernatant was harvested, filtered (0.45 μm filter, Sartorius) and usedto infect new uninfected SW626 cells plated at the same density asbefore. Samples of virus supernatant were collected routinely and storedat −70° C. MLV RT assays and hence viral titres were determined every 14days in order assess the growth pattern of the virus. This pattern ofinfecting new SW626 cells was maintained continuously until there wasevidence of a new MOLV4070A variant (FIG. 6).

In FIG. 6 it appeared that by day 34 a new MOLV4070A variant populationhad emerged, this variant was designated MLV626-1. The new variantpopulation was grown for a further 41 days using the same passagingprotocol. The resulting population was designated MLV626-2. PopulationsMLV626-0, MLV626-1 and MLV626-2 were then compared by infecting newuninfected SW626 cells and monitoring growth patterns over a 5 dayperiod. MLV RT assays were performed and the viral titres calculated.The results are shown in FIG. 7. It appears that the new variantMLV626-1 infected SW626 cells more efficiently than the starter virusMLV626-0 with viral titres of 1.2×10⁴ for MLV626-1 and 3.9×10³ forMLV626-0 on day 5. Interestingly, however, the virus MLV626-2 had aviral titre of 2.4×10⁴ which was higher than the titre of MLV626-1. Itappears that MLV626-1 and MLV626-2 are in fact new variants of MOLV4070Aand that these variants have the ability to infect SW626 cells moreefficiently than the starter virus MLV626-0.

SW626 cells infected with either MLV626-1 or MLV626-2 were diluted sothat 10 cell populations were isolated in a well of a forty-eight wellplate and allowed to grow to 80%-85% confluence. A MLV RT assay wasperformed on virus supernatant from each isolated SW626 cellularpopulation. Products of the reaction were spotted onto DE81 paper andexamined by autoradiography. The MLV626-2 clones producing the strongestsignals were selected. These clones were then expanded and total DNAprepared according to the method of Blin and Stafford (Nucl. Acids. Res3, 2303.1976). This DNA was then used as a template for two PCRreactions. The first reaction, carried out using Advantage PCR kit(Clontech), used a 5′ primer CGCGGATCCGMTTCATGGGCCAGACTGTTACCACTCCC [SEQID NO: 1] and a 3′ primer CGCGTCGACTCTAGATTAGGGGGCCTCGCGGGTTTMCCTTA [SEQID NO: 2] and produced a fragment of 5.2kb comprising the gag-polcassette. The second reaction, again using the Advantage PCR kit, used a5′ primer CGCGCTAGCTCTAGMTGGCGCGTTCAACGCTCTCAAAA [SEQ ID NO: 3] and a 3′primer CGCGGATCCTCATGGCTCGTACTCTATGGGTTT [SEQ ID NO: 4] and produced a 2kb fragment comprising the amphotropic env cassette. The 5.2 kb fragmentwas cut with EcoRi and Sall and inserted into pCIneo (Promega) toproduce a gag-pol expression plasmid designated pE6262 and the 2 kbfragment was cut with Nhel and EcoRi and also inserted into pCIneo toproduce and env expression plasmid designated pGP6262.

Plasmids pE6262 and pGP6262 were then used as the gag-pol and envexpression plasmids in a 3-plasmid transfection system to produce lacZtransducing retroviral particles (Soneoka et al).

Example 3

Many metabolic deficiencies are the result of low or absent levels ofproteins in the liver. Efficient gene transfer to the liver is animportant aspect, therefore, of future gene therapy strategies. Usingsimilar strategies to those used in Examples 1 and 2 we produce a TORVfor hepatocytes. This will increase the ability to deliver genes to theliver.

Example 4

Murine retroviruses are known to be inactivated by complement componentspresent in human serum (Takeuchi et al., 1994).

This seriously limits the ability to use MLV-based retrovirus vectors invivo. This is known to be partially alleviated by preparation of thevector particles in non-murine cells (James Respess, pers. comm.;Takeuchi et al., 1994) although the particles are still somewhatsensitive. Using similar strategies to those used in Examples 1 and 2 weproduce a retroviral vector system that has increased resistance toinactivation by human complement. Instead of using target cell types forthe selection stage we use exposure to human serum. In particular hightitre retrovirus preparations produced in either murine 3T3 cells orHeLa cells or any other cells type are exposed to differentconcentrations of human serum. Concentrations are from neat to a{fraction (1/1000)} dilution in buffer (Takeuchi et al., 1994). After 2hours exposure, and therefore selection, remaining virus is plated ontofresh cells in order to allow amplification of the selected population.This represents one round of selection. Many rounds of selection arecarried out until the maximum resistance to exposure to human serum isachieved. As with Example 1 several parallel variant populations will beselected and then combined in order to allow for recombination. Highlyresistant MLV variants will then be used to produce new vector systemsas described in Examples 1 and 2. The resulting vector system hasgreater utility for human gene therapy particularly for those protocolsthat require delivery in vivo.

Example 5

In many gene therapy situations high titre transducing virus stocks arerequired. In order to achieve these high titres it is necessary toconcentrate the preparation. However, retroviruses are susceptible tothe shear forces that occur in most centrifugation or filtration methods(see Burns et al., 1993)). This means that while virus particles areconcentrated many of the particles lose their envelope glycoproteins andso the effective transducing titre is reduced. Using similar strategiesto those used in Examples 1 and 2 we produce a retroviral vector systemthat has increased resistance to inactivation by shear forces. In thisexample selection is imposed on the population by exposing the virus tothe shear forces inherent in passing through a gauge 10 syringe needle.Instead of using target cell types for the selection stage we usepassage through the syringe needle. In particular, high titre retroviruspreparations produced in either murine 3T3 cells or HeLa cells or anyother cell type are passed through a gauge 10 syringe needle from a 5ml.syringe. The remaining virus is plated onto fresh cells in order toallow amplification of the selected population. This represents oneround of selection. Many rounds of selection are carried out until themaximum resistance to shearing is achieved. As with Example 1 severalparallel variant populations will be selected and then combined in orderto allow for recombination. Highly resistant MLV variants will then beused to produce new vector systems as described in Examples 1 and 2. Theresulting vector system has greater utility for human gene therapybecause higher titres can be achieved by concentration by centrifugationor filtration.

Example 6

A long standing goal of retroviral vector design has been thedevelopment of targeted vectors that are able to transduce specific celltypes. The advantages of targeted vectors are that vector particles arenot ‘wasted’ on non-target cells and it may be possible to increase theefficiency of gene transfer following administration of the vectors invivo. A common strategy that has been employed by several laboratorieshas been the use of vectors bearing manipulated surface proteins. Theseproteins are generally hybrid molecules comprising the retroviralsurface protein backbone with a sequence of amino acids inserted thatbinds to a specific cell surface molecule on the target cell. Severaltargeting systems have been produced and these include EPO (Kasahara etal. 1995), single chain antibodies (Somia et al. 1995; Chu and Dornberg1995), growth factor-like sequences (Han et al. 1995; Cosset et al.1995) and integrin-binding peptides (Valsesia-Wittman et al. 1994). Insome of these cases transduction of the specific target cell isachieved. However, in many of these cases efficiencies are low and thereis a need for the inclusion of the wild-type surface protein in thetargeted particles. This makes them of very limited use in therapeuticapplications.

It is possible to increase the efficiency of these manipulated istargeted systems by applying the principles described in the previousexamples. If the manipulated surface protein gene is incorporated into areplication competent provirus then increased efficiency can be selectedby passaging the virus on the target cells. Alternatively, a manipulatedsurface protein gene can be incorporated into a retroviral vector genomeand increased efficiency selected by component specific evolution (FIG.1).

Using these strategies, for example, in the system described by Han etal. (1995) it is possible to substantially increase the efficiency ofgene delivery to breast cancer cells by retroviral vector particlesbearing a hybrid surface protein containing heregulin. Replicationcompetent retroviruses in which the natural surface protein gene isreplaced by the heregulin hybrid are passaged on MDA-MB453 or otherbreast cancer cell lines. These cell lines could include the gene forwild-type ecotropic envelope proteins in the early stages of selectionin order to facilitate infection of the target cells. Later in theselection scheme it may be possible to use unmanipulated cells if theselection process had yielded virus that no longer requires thewild-type surface protein to infect the target cells. When vectorsystems are produced from these selected viruses they transduce breastcancer cells at efficiencies that make them clinically useful for thetreating of breast cancer by gene therapy.

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4 1 39 DNA Synthetic primer 1 cgcggatccg aattcatggg ccagactgtt accactccc39 2 42 DNA Synthetic primer 2 cgcgtcgact ctagattagg gggcctcgcgggtttaacct ta 42 3 39 DNA Synthetic primer 3 cgcgctagct ctagaatggcgcgttcaacg ctctcaaaa 39 4 34 DNA Synthetic primer 4 cgcggatcctcatggctcgt actctatggg tttt 34

What is claimed is:
 1. A method of making a retroviral vector having oneor more of the following selected characteristics: an increased abilityto infect a chosen target cell, or increased resistance to shear forcesor increased resistance to human serum, which method comprises: (i)providing a starting retrovirus or retroviral vector, and a host cellfor the retrovirus or the retroviral vector; (ii) subjecting thestarting retrovirus or retroviral vector to a selection process in vitrowhich selection process involves a plurality of rounds of infection ofthe host cell during which the retrovirus or retroviral vector evolvesto attain the selected characteristics; and (iii) where a startingretrovirus is provided in (i), the retrovirus resulting from (ii) isused in at least one component of a retroviral vector production systemfor producing retroviral vectors having the selected characteristic orcharacteristics.
 2. A method as claimed in claim 1, wherein the hostcell is a chosen target cell and the selected characteristic is anincreased ability to infect the target cell.
 3. A method as claimed inclaim 1, wherein the selection process involves exposing the retrovirusor retroviral vector to shear forces or human serum between rounds ofinfection, and the selected characteristic is an increased resistance toshear forces or an increased resistance to human serum, respectively. 4.A method as claimed in claim 1, wherein the selected characteristic is aproperty or a packaging component or components of the retrovirus or theretroviral vector.
 5. A method as claimed in claim 1, wherein theretrovirus or retroviral vector is, or is derived from, MLV.
 6. A methodas claimed in claim 1, for making a retroviral vector for use in genetherapy.
 7. A method as claimed in claim 1, wherein the startingretrovirus or retroviral vector comprises a selectable marker.
 8. Anevolved retroviral vector made by the method according to claim 1,wherein the retroviral vector has an increased ability to infect achosen target cell and/or increased resistance to shear forces.
 9. Anevolved retroviral vector production system, said system having at leastone component having a selected characteristic or characteristicsattained by the method according to claim 1 and transferred from theretrovirus or retroviral vector into the retroviral vector productionsystem said component conferring upon the retroviral vector an increasedability to infect a chosen target cell and/or increased resistance toshear forces.
 10. An expression vector comprising a packaging componenthaving a selected characteristic of a retrovirus or a retroviral vectorobtained by the selection process (ii) of the method according to claim1 and having an increased ability to infect a chosen target cell and/orincreased resistance to shear forces.
 11. An expression vector encodinga genome of a retroviral vector, which genome is derived from the genomeof a retrovirus or retroviral vector resulting from the selectionprocess (ii) of the method according to claim 1 and having an increasedability to infect a chosen target cell and/or increased resistance toshear forces.
 12. A method as claimed in claim 2, wherein the startingretrovirus or retroviral vector comprises a manipulated surface proteingene and the selected characteristic is an increased ability to infectthe target cell via the surface protein encoded by the manipulatedsurface protein gene.
 13. A method as claimed in claim 2, wherein humanlymphocytes or hepatocytes or cancer cells are the chosen target cell.14. A method as claimed in claim 4, wherein a starting retroviral vectoris used which comprises a selectable marker and the packaging componentor components.
 15. A method as claimed in claim 4, wherein the packagingcomponent is env.
 16. A method as claimed in claim 4, wherein thepackaging components are env and gag-pol.
 17. A method as claimed inclaim 14, wherein the packaging component or components of theretroviral vector resulting from the selection process (ii) is or areused in a retroviral vector production system to produce retroviralvectors having the selected characteristics.
 18. An evolved retroviralvector as claimed in claim 8, having a genome containing atherapeutically active gene.
 19. An evolved retroviral vector productionsystem as claimed in claim 9, comprising a packaging cell linetransfected with a DNA construct encoding a packagable retroviral vectorgenome.
 20. A packaging cell line of a system according to claim 19,having a selected characteristic associated with it.